The present invention concerns methods and apparatus for measuring or identifying the presence of selected substances in the body and/or assessing blood brain dynamics of a subject via non-invasive spectrographic analysis of certain regions of the eye, such as the aqueous humor in the anterior chamber of the eye.
Non-invasive measurement of physiological and foreign substances, including blood glucose, by optical spectroscopy techniques has remained an elusive target for at least two decades. Blood, tissue, and most excreted fluids contain numerous substances which confound many spectral signatures. On the other hand, the aqueous humor (AH), which fills the anterior chamber of the eye (between the lens and cornea), contains relatively few molecules capable of interfering with the spectroscopic detection of glucose. These are primarily lactate, ascorbate, and urea. This fact, along with its optically accessible location behind the cornea, makes the AH an attractive choice as a site on which to attempt non-invasive analysis of many substances present in a biological subject, including glucose.
Pohjola (Acta Ophthalmologica Suppl. 88, 1-80 (1996)) showed that the ratio of aqueous glucose to plasma glucose in normal euglycemic individuals is related to age and ranges from 0.6 to 0.9. He further showed in seven humans with steady-state hyperglycemia that similar ratios applied. There is little, if any, data regarding the equilibration time of aqueous humor glucose with changes in plasma glucose in humans. Some recent research suggests that the glucose content of the AH compared with that in the capillary blood in man is about 0.75 regardless of the glycemic state of the person. See e.g., Schrader et al., The glucose content of the aqueous humour compared with capillary blood in man, Invest. Ophthalmol. Vis. Sci. (Suppl.) 44:404 (2000).
Numerous investigators over the years have suggested that the ratio of aqueous glucose to plasma glucose in the normoglycemic rabbit ranges from 0.42 to 1.01 (S. Pohjola, supra; D. Reddy and V. Kinsey, Arch. Ophthalmol. 63, 715-720 (1960); M. Reim et al., Ophthalmologica 154, 39-50 (1967); W. March et al., Diabetes Care 5, 259 (1982)). It is uncertain whether this variability is normal or could be attributed to differences in glucose measurement techniques, collection techniques, sample storage, and anesthesia. It is believed that the relationship of aqueous glucose to rising, or falling, plasma glucose has not been previously studied in rabbits.
Cotxc3xa9 has reviewed the relative strengths and weaknesses of optical glucose sensing techniques (J. Clin. Engineering 22, 253 (1997)). Raman spectroscopy is potentially attractive because it can distinguish glucose in water solutions containing various levels of other optically active metabolites (S. Wang et al., Applied Optics 32, 925 (1993)). Raman spectroscopy measures the shift in the wavelength of incident light as it is scattered by molecules. Any given molecule typically causes a characteristic shift in the spectrum of scattered light, which is dependent upon its intermolecular and intramolecular bonds. This is in contradistinction to fluorescence, which is caused by changes in electron energy states, and does not shift relative to the wavelength of incident light.
Wicksted et al, (Appl. Sectroscop. 49, 987 (1995)) suggest that the Raman signature for glucose can be identified in aqueous humor samples, and Goetz et al. (IEEE Trans. Biomed. Eng. 42, 728 (1995)) have demonstrated that higher than physiologic levels of glucose can be measured with Raman spectroscopy in water solutions. J. Lambert et al. (LEOS Newsletter 12, 19-22 (1998)) suggest that measurement of glucose at physiologic levels is possible in water solutions containing other analytes normally found in the aqueous humor. In certain situations, when solutions containing fluorescent substances are studied, however, the fluorescence signal may overwhelm the relatively weak Raman-shifted signal. This is a potential problem if Raman spectroscopy is applied to certain regions in the eye, such as the conjunctiva or vitreous or aqueous humor (and/or depending upon what the Raman signal is attempting to identify or measurer), which can contain proteins that fluoresce.
U.S. Pat. No. 5,243,983 to Tarr et al. proposes a non-invasive blood glucose measurement system using stimulated Raman spectroscopy. Stimulated Raman spectroscopy can require the use of both a pump and a probe laser beam. In operation, the probe laser beam is used to measure the stimulated Raman light at a single wavelength after transmission across the anterior chamber of the eye. Commercially, this may be undesirable, since an optical component contacting the eye is used to direct the beam across the anterior chamber. In addition, use of a single wavelength may limit the ability to measure glucose at physiologic levels within tissue containing many other Raman scattering chemicals.
Others have also proposed various glucose measurement devices. For example, U.S. Pat. No. 5,433,197 to Stark suggests a non-invasive glucose measurement apparatus that employs broadband, infrared light stimulation. In addition, U.S. Pat. No. 5,553,617 to Barkenhagen proposes a non-invasive method for measuring body chemistry from the eye of a subject by measuring a spectral response such as a Raman scattering response. While the latter reference alleges that it may be used for medical applications (such as the determination of sugar in diabetics), specific details on how this might be accurately carried out are not provided. Another example is found in U.S. Pat. No. 5,710,30 to Essenpreis, which proposes a method for measuring the concentration of glucose in a biological sample such as the eye (see FIG. 4 therein) with interferometric measurement procedures. Still another example is proposed in U.S. Pat. No. 5,666,956 to Buchert et al., wherein it is proposed that an instrument for the non-invasive measurement of a body analyte can be based on naturally emitted infrared radiation.
In spite of the foregoing efforts, a commercially viable, non-invasive monitor which can successfully employ a non-invasive optical analysis of certain regions of the eye, including the aqueous humor of the eye, has not yet been developed. Difficulties in developing such a device include: (a) determining reliable correlations of the typical millimolar quantities of selected substances or chemicals; (b) obtaining accurate measurements of selected substances; and (c) inhibiting damaging effects to the eye which may be caused by excessive exposure to light in an instrument that is used to generate the analysis signal spectrum in the AH. Accordingly, there is a continued need for improved systems, methods, and devices for the non-invasive in vivo analysis of foreign and natural physiologic substances in a biological subject via optical analysis of certain regions of the eye.
The present invention provides methods and systems for monitoring or evaluating the blood aqueous barrier and, thus, the blood brain barrier dynamics of the subject. The present invention also provides methods which can detect the presence or absence of one or more selected substances or analytes of interest in the body by optically analyzing certain regions of the eye, including at least one of the AH, the vitreous humor (xe2x80x9cVHxe2x80x9d), and one or more blood vessels in the conjunctiva. In certain embodiments, the analysis can provide information regarding the presence of and/or quantify a detected substance in the cerebral spinal or intracranial fluid of the subject (indirectly, through a correlation with the presence or quantification of the substance in the blood vessel in the conjunctiva, or the AH or the vitreous humor).
Embodiments of the invention can employ Raman spectroscopy to non-invasively obtain, in vivo, at least one signature spectroscopic signal to identify and/or measure the level or concentration of a substance or substances of interest in the subject (either in the blood and/or brain) based on the signal.
In certain embodiments, the present invention can be used to monitor or evaluate the blood brain barrier dynamics, which may be intentionally altered (such as through the administration of chemicals or exposure to certain environmental conditions such as increased pressure) during such evaluation. By intentionally breaking down the blood brain barrier, medicines which are normally inhibited from crossing the barrier may be allowed to more readily cross and enter into the brain. To monitor such a change in the operation or dynamics of the blood brain barrier, non-specific markers can be introduced or injected into the subject. The non-specific marker is selected based on its molecular size and/or its normal reluctance to cross the blood brain barrier. The blood brain barrier can then be intentionally altered (broken down or opened) so that the non-specific marker is able to cross therethrough. An optical reading of a selected region of the eye can be taken, and the present invention can assess whether the marker is present (either at all or in an increased amount over a pre-alteration state) in the blood aqueous barrier. Further, in some embodiments, the concentration of the marker in the eye can be determined (such as in the AH or vitreous humor) in the blood aqueous compartment. If the marker is identified as being present, this indicates that the blood brain barrier has been altered. Once the blood brain barrier is altered, a desired treatment regimen can be administered to the subject (such as drug used for chemotherapy) to treat tumors or other conditions in the eyes or brain. In some embodiments, as an alternative to the use of non-specific markers, the present invention can monitor the presence or concentration of the treatment drug itself in the blood aqueous compartment in the eye. Examples of suitable markers include large molecule natural and synthetic substances which do not normally cross the blood brain barrier, including, but not limited to, antibiotics such as erythromycin, and conjugated substances such as conjugated billirubin.
Typically, the treatment drug is configured such that it is inhibited from crossing the blood brain barrier. Thus, in certain applications, in order to deliver a sufficient quantity of the drug to the brain, the amount of drug, which is systemically delivered, can be undesirably toxic to the patient away from the targeted treatment region in the brain. The present invention can now assess or assure either that the barrier is sufficiently altered to allow the drug to pass more efficiently therethrough and/or quantify or assess that a sufficient amount of the treatment drug is getting into the brain so that the systemic amount can be more closely regulated and reduced. After the desired treatment (or dose) is indicated as delivered to the brain, the blood brain barrier can be restored such that it is substantially in its pre-altered state. The return to the pre-altered state can be confirmed by taking another optical reading to confirm that the marker is in a reduced concentration in the blood aqueous compartment in the eye.
By identifying and/or quantifying the amount of the non-specific marker present in the aqueous humor (and, thus in the blood aqueous barrier), an estimate or determination of the concentration in the brain can be established. Typically, the concentration in the blood aqueous humor can be presumed to be similar to that in the blood brain compartment. Further, the two concentrations can be correlated so that a quantitative value of the amount in the cerebral spinal or intracranial fluid can be determined based on that found in the blood aqueous compartment so that a titrated dose of the treatment drug in the brain may be able to be determined. The correlation relationship or ratio may vary within certain population segments. In certain embodiments, the correlation relationship can be determined based on data collected across a representative population (by age, size, weight, gender, race, disease or physiological impairment or abnormality, or national origin). Thus, the amount of the selected treatment drug actually getting into the brain can be correlated to the systemic dose delivered to the patient so that the dose or level in the spinal fluid is sufficiently high for treatment of the tumor or other condition but the systemic dose is sized to provide reduced toxicity to the subject (by avoiding administering unnecessary quantities to the subject).
In certain embodiments, the present invention can provide methods, similar to that discussed for intentionally altering the dynamics of the blood brain barrier, which use a non-specific marker with a new drug to evaluate the impact that a new drug or therapy regimen has on the blood brain barrier for safety or other considerations. In other embodiments, the optical detection of the drug itself in certain regions of the eye, without the use of a marker, may be sufficient to indicate the drug""s impact on the blood brain barrier.
In other embodiments, the environmental conditions surrounding the patient or subject, can be altered and the dynamics of the blood brain barrier monitored. For example, subjects which are exposed to different elevations, gravity conditions, or to increased intracranial pressure, may exhibit different or altered blood brain barrier characteristics, either transiently, or more chronically, than persons not so exposed. These subjects may include astronauts, pilots, divers, trauma victims, and the like. Evaluating the blood brain barrier dynamics can identify whether larger molecules or pathogens are able to cross the blood brain barrier, which may, under normal circumstances, be inhibited or prevented from entering the brain. In certain embodiments, the present invention can be used to assess which constituents in the blood cross into the intracranial fluid via the blood brain barrier.
As generally described above, in one embodiment, a patient can undergo a treatment regimen to deliberately or intentionally alter the blood brain barrier dynamics so that an identified treatment agent(s) is allowed to cross the barrier. For example, an osmotic agent such as a drug (for example, MANATOL) can be delivered to a subject being treated for cancer to force the blood brain barrier to open (preferably for a limited-time treatment window) to successfully allow a selected chemotherapeutic agent (such as a cytotoxic agent) to be able to more readily cross the blood brain barrier into the brain. Non-invasive monitoring of the tumor dose according to the present invention, can allow monitoring of the barrier dynamics and may, in some embodiments, be able to assess when an adequate, but not excessive, tumor dose is delivered to the brain. The blood brain barrier can be reestablished after the appropriate tumor dosing is delivered. This monitoring of the blood brain barrier dynamics during a treatment regimen may now inhibit or reduce systemic damage in the subject associated with the cytotoxic treatment.
In other embodiments, the present invention provides systems and methods for detecting the presence of a predetermined substance or identifying the presence of an unknown substance in the body of a subject. The substances which can be measured or identified are numerous and can be (a) natural physiologic analytes or chemicals, such as glucose, amino acids, peptides, antibodies, blood (typically using light outside the red spectrum), and/or (b) foreign substances such as medicaments, drugs, or poisons (whether legal or illegal, and whether prescription or over the counter). For example, the present invention can be used to assess the presence of targeted illegal substances, such as alcohol or illegal narcotics such as cocaine, pcp, marijuana, or to identify what toxin or poison a subject has injested out of a number of household or environmental toxins and/or poisons such as herbicides, pesticides, household cleaning products, petroleum products or other common house hold chemicals including benzene, ethylene glycol. The methods and systems of the instant invention may even be used to identify the presence of poisonous plants, insect toxins, and reptile or snake venom. The present invention may be configured to identify whether an unknown substance in a subject is one or more of toxins/agents associated with the most prevalent poison-related emergency room visits. For example, ethylene glycol, methanol, and acetaminophen.
In certain embodiments, the present invention can be used to quantify the amount of the substance in the subject, typically this embodiment may be particularly suitable for those substances ingested in relatively large quantities or those present in sufficient quantity in the selected region of the eye so as to be detectable in vivo, or so that the substance or analyte is present in physiological levels (in the blood or blood aqueous compartment) of above about 0.001% or above about 1-10 xcexcmolars, depending on the Raman active characteristics of the analyte of interest.
In addition, in certain embodiments, the devices and methods of the present invention may be used to detect increased or decreased levels of physiologic analytes such as caused by system impairments or reactions associated with dehydration, allergic reactions, or physiologic analytes associated with bacterial infections such as spinal meningitis, or to identify whether proteins or antibodies are present in elevated levels to identify a systemic response or a localized infection or disease in the eye or an immune system response, in the subject.
In certain embodiments, the systems and methods of the present invention may be able to detect or identify toxins released or emitted from foods contaminated with food poisoning bacteria such as E coli, salmonella (either in vivo or in vitro). Further, in some embodiments, the methods and systems of the present invent may be used to identify the presence of mad cow disease by analyzing certain regions of the eye (either in vitro or in vivo) such as by obtaining a Raman spectrum of a desired region of the eye analyzing the spectrum to detect the presence of small peptides or other markers associated with the disease. Other diseases may be able to be identified in vivo by the presence of a systemic reaction (such as an increased constituent level of a natural physiologic substance) in the subject. It is anticipated that such a method may be potentially used to assess whether the subject has contracted Lyme disease associated with deer tick bites or Rocky Mountain spotted fever.
In some embodiments, the invention can identify the presence of one or a plurality of household or environmental poisons in the subject in a relatively fast xe2x80x9ctriagexe2x80x9d assessment to allow clinicians to determine the appropriate treatment in a timely manner. This can be particularly important for pediatric applications where the substance ingested may be difficult to ascertain for young children, and a relatively quick identification of a particular toxin or toxins ingested may allow more reliable or faster treatment decisions to be established.
One embodiment of the invention is directed to an in vivo method for monitoring the blood brain barrier dynamics of a subject, comprising the step of monitoring the dynamics of the blood brain barrier by non-invasively obtaining the Raman spectrum of a selected region in the eye of the subject. The method may also include the step administering a non-specific marker to the subject selected for its normal reluctance to cross the blood brain barrier under the normal condition. The monitoring step can comprise detecting the presence of the non-specific marker in the selected region of the eye of the subject.
In certain embodiments, the method can include the steps of: altering the dynamics of the blood brain barrier of the subject from a normal condition; and administering a quantity of a selected therapeutic agent to a subject for treatment of condition in the brain or neurological system after the altering step. It can also include the step of substantially returning the blood brain barrier to its normal state after a sufficient quantity of the therapeutic agent has been delivered to the brain. Similarly, the monitoring step can be performed before the therapeutic drug is administered to the subject and subsequently to confirm that the blood brain barrier is substantially returned to its normal condition. The method may also include the step of assessing the dose amount of the therapeutic agent delivered to the brain.
Certain embodiments of the present invention are directed to an in vivo a non-invasive method for determining the level of an analyte of interest in a biological subject. Raman spectroscopy can be used to obtain the signature of the substance in the eye (such as in the AH, VH, or blood vessel in the conjunctiva) and, in some embodiments, to measure the concentration of a natural physiologic or foreign substance, such as glucose and/or proteins, or drugs, alcohol, environmental or household toxins, in the subject. The method can include the steps of: (a) generating an excitation laser beam (e.g., at a wavelength of from about 600 to 900 nanometers); (b) focusing the excitation laser beam into the eye of the subject so that a selected region of the eye is illuminated; (c) detecting (preferably confocally detecting) a Raman spectrum from the illuminated region of the eye; (d) comparing the Raman spectrum from the detecting step to predetermined spectrums corresponding to different analytes of different concentrations; and (e) identifying the presence of an analyte of interest based on the detecting and comparing steps.
In some embodiments, an additional step (f) can be performed to determine the blood or brain level of an analyte of interest for the subject from the Raman spectrum. The blood or brain level may be indirectly computated based on the concentration or amount of the analyte in the blood aqueous compartment (or can be directly measured in the blood itself for the conjunctiva vessel measurement). For the indirect measurement, that value can be correlated (or adjusted/corrected) to provide an assessment of the amount of the substance in the cerebral spinal fluid or blood. The correlation may be such that the amount of the substance directly measured in the AH is substantially similar to that in the cerebral spinal fluid. Alternatively, data correlating the relationship can be established and an empirical or statistical model established.
Although not required, in some embodiments, the detecting step can be followed by the step of subtracting a confounding fluorescence spectrum from the Raman spectrum to produce a difference spectrum; and determining the blood level of the analyte of interest for the subject from that difference spectrum, preferably using linear or nonlinear multivariate analysis such as partial least squares or artificial neural network algorithms. This technique may be particularly suitable where fluorescence is problematic for optical measurements taken directly of the blood level (i.e., by focusing at the blood vessels in the conjunctiva or at the vitreous humor).
In certain embodiments, a low energy excitation wave can be used to generate the Raman signal spectrum. xe2x80x9cLow energyxe2x80x9d, as used herein, means power which is on the order of about 10-400 mJ or less, and typically between about 70-330 mJ. The energy exposure will depend on the power and pulse length of the excitation pulse. Longer wavelength pulses (i.e., above 700) may be used, typically with energy levels closer to the higher end of the scale, while lower wavelengths (600-700) may be used with lower energy exposure levels. In one embodiment, a wavelength of about 633 nm can be used for a pulse of about 5-10 seconds corresponding to about a 2-5 mW power exposure level (and between a 10-20 or 25-50 mJ energy exposure to the patient""s eye (or eyes)) for each measurement or monitoring signal obtained. In other embodiments, an optical excitation pulse may have a 785 nm wavelength, a pulse length of about 20 ms-5 s and a power rating of about 14-16 mW. In one embodiment, a 5 sec, 16 mW pulse can be used to obtain the in vivo reading of a cancer agent in the selected region of the eye (typically the AH).
In some embodiments, the excitation beam can be transmitted such that it presents a reduced energy/density exposure rating to the tissue of the eye by shaping the beam to increase the cone angle or span of the excitation beam as it enters the eye to expose more of the area of the retina and reduce the energy/area rating of the excitation pulse to provide improved margins of safety (placing the energy/area rating sufficiently below the threshold of damage). In other embodiments, the transmission path numerical aperture is substantially matched to the return path numerical aperture (of the spectrometer).
A second aspect of the present invention is an apparatus for the non-invasive in vivo determination of the blood level of an analyte of interest in a subject. The apparatus includes a laser source for generating an excitation laser beam (e.g., at a wavelength of from about 600 to 900 nanometers) and an optical system (e.g., a confocal optical system) operatively associated with the laser for focusing the excitation laser beam into a selected region of the eye, including one or more blood vessels in the conjunctiva of the eye, the vitreous humor, or the anterior chamber of an eye (or eyes) of the subject so that the aqueous humor in the desired region of the eye is sufficiently illuminated. The apparatus also includes a detector operatively associated with the optical system and configured to detect a Raman spectrum from the selected illuminated region of the eye and a processor with computer program code for identifying the presence of one or more selected substances or analytes of interest. The computer code may also include code for determining the in vivo level of the analyte of interest in the selected region of the eye and to establish an estimate or measure of the analyte in the blood or cerebral spinal fluid to be established based on a correlation thereto for the subject from the Raman spectrum.
Focusing the optical analysis on the blood vessels in the conjunctiva can allow for a direct measurement of the substance in the blood, while the measurements taken from other portions of the eye can be correlated to provide an estimate or quantification of the substance in the blood and/or in the cerebral spinal fluid (i.e., indirect measurements). The correlation""s can be established based on empirical models or actual measurements taken in vitro or in vivo on a representative animal or human population as is well known to those of skill in the art.
In certain embodiments, the apparatus can be configured as a low energy unit to inhibit the exposure of the tissue during the operation of the apparatus. The excitation wavelength at the low power may be less than 700 nm, such as about 633 nm.
Numerous additional features may be incorporated into the apparatus. The device may include a visual display screen for presenting visual indicia to the user, which can be individually adjusted and focused to the particular visual acuity of the subject (similar to vision screening focusing procedures). The apparatus may include a visual display screen for visually displaying the results of the test to the subject (such as through the same aperture or adjacent active matrix screen) as which the test is conducted. It may include a visual fixation target or device, also visible through the test aperture, which controls movement of the eye and simultaneously insures that focusing of the laser beam is properly directed into the anterior chamber of the eye. The processor may contain empirical models of actual testing experience to either determine the blood level or concentration of the analyte of interest or to identify the presence of selected substances. The apparatus may employ a laser of fixed wavelength, a tunable laser (which can sample a plurality of Raman scattered light (at different wavelengths) concurrently), a plurality of fixed wavelength lasers, or other light source means some of which can include means for sliding the Raman spectrum passed a plurality of different wavelength detectors to obviate the need for a full grating based Raman spectrometer (by taking a plurality of samples). The apparatus may include a wireless or remote communication line operably associated with the processor for transmitting the blood level of the analyte of interest to a remote location (such as for emergency home calls to an ER room).
Another aspect of the present invention is an in vivo method for administering drug or chemical therapy to a subject (such as for treatment of a cancerous tumor in the brain). The method includes the steps of: (a) administering a dose of a selected therapeutic agent to a subject; (b) altering the dynamics of the blood brain barrier from its normal state; (c) monitoring the dynamics of the blood brain barrier by non-invasively obtaining the Raman spectrum of a selected region in the eye (such as the vitreous or aqueous humor) and determining the quantity of the agent therein. The method may also include one or more of (d) estimating the dose of the therapeutic agent delivered to the brain (indirectly, based on the amount detected in the selected region of the eye) (e) repeating said monitoring step a plurality of times during the administering step; and (f) returning the blood brain barrier to its normal state after a sufficient quantity of agent has been delivered to the brain.
In one embodiment, the altering step can be carried out by introducing a chemical to the subject to temporarily open the blood brain barrier to allow larger molecules to pass therethrough. Further, the method can include the step of administering a non-specific marker which is reluctant to or does not normally pass through the blood brain barrier (i.e., is typically inhibited from passing therethrough). The optical analysis can monitor any increase (or the presence) of the non-specific marker in the selected region of the eye to confirm that the blood brain barrier dynamics has been altered.
In another embodiment, the altering step may be carried out by increasing the intracranial pressure of the subject.
Another aspect of the present invention is a method of non-invasively monitoring the blood brain barrier. The method comprises the steps of: (a) generating an excitation beam at a wavelength of from 600 to 900 nanometers; (b) focusing the excitation beam of said generating step into the anterior chamber of an eye of the subject so that aqueous humor in the anterior chamber is illuminated; (c) detecting a Raman spectrum corresponding to the illuminated aqueous humor; and (d) monitoring the AH to predict the behavior of the blood brain barrier dynamics during exposure to selected conditions based on the detecting step (based on the AH Raman spectrum analysis"" indication of the presence or concentration of selected substances therein). It is anticipated that the correlation between the blood-aqueous and blood brain barrier is such that the presence and/or concentration in one can be extrapolated to that in the other.
Other embodiments focus the excitation beam such that it has an increased or wider cone angle to spread the light across more area of the retina. Still other embodiments are configured to focus to one or more blood vessels on the conjunctiva or to focus deeper to the vitreous humor.
In certain embodiments, the monitoring step can be used to assess whether the dynamics thereof alter sufficiently to allow selected analytes, which would normally be inhibited from traveling through the blood brain barrier, to pass into the intracranial spinal fluid through the blood brain barrier. In other embodiments, the monitoring step can be carried out when a person is under or exposed to extreme conditions such as when diving, flying, or mountain climbing, or when suffering from a traumatic head or brain injury, or high stress, and the like.
The method can also include the steps of comparing the Raman spectrum from the detecting step to reference spectrums corresponding to at least one selected analyte of interest; and identifying the presence of the least one analyte of interest in the subject based on the detecting and comparing steps in the selected region of the eye. The method may also be able to estimate the dose or affirm the presence of the analyte in the subject""s cerebral spinal fluid.
Yet another aspect of the present invention is a method for identifying an alteration in the blood brain barrier of a biological subject, comprising the steps of: (a) non-invasively obtaining a first in vivo Raman spectrum of the aqueous humor of the subject; (b) non-invasively obtaining a second in vivo Raman spectrum of the aqueous humor of the subject; and (c) identifying an alteration in the blood brain barrier by comparing the first and second Raman spectrums.
An additional aspect of the present invention is a method for identifying an alteration in the blood brain barrier of a biological subject, comprising the steps of: (a) non-invasively obtaining a first in vivo Raman spectrum of the aqueous humor of the subject; (b) obtaining a reference spectrum of an in vitro sample representing the aqueous humor and comprising at least one selected analyte; and (c) comparing the in vivo Raman spectrum to the reference spectrum to identify an abnormality in the blood brain barrier by detecting the presence of at least one selected analyte in the AH thereby indicating its presence in the intracranial fluid of the subject.
The at least one selected analyte can be one which typically does not cross the blood brain barrier so that its presence is indicative of an abnormality or impairment or successful intentional alteration of the blood brain barrier dynamics.
Each of the embodiments of the invention may include computer program products and computational and look-up table associated therewith to identify the presence of the selected substance or substances of interest (and/or calculate the amount or concentration thereof) and to operate or control (regulate) the power of the excitation pulse emitted from the laser, and the illumination and detection of the scattered light. For example, in certain embodiments, the present invention can include a computer program product for determining the identity of an unknown substance in a subject. The product can comprise computer-readable program code comprising: (a) computer readable program code for defining at least one signature reference spectrum for at least one selected substance of interest; (b) computer readable program code for analyzing an in vivo obtained Raman spectrum of the aqueous humor of the subject; and (c) computer readable program code for based evaluating whether the in vivo Raman spectrum corresponds to at least one of the at lest one signature reference spectrums by comparing selected characteristics between the reference spectrum and the in vivo spectrum.
In various embodiments, the computer readable program code for defining the different reference spectrums can be for a particular one or a plurality of different selected substances. Examples of the selected substance(s) include, but are not limited to: alcohol, a substance banned for athletes in competition, a plurality of illegal narcotic substances, and a plurality of household products or common poisons for humans or animals which are potentially poisonous to a subject when ingested. A master look-up reference data base providing Raman spectrum data for a large quantity of different poisons or substances can be generated and stored at a central database or at local or regional offices, clinics or the like. The computer program can include means for remotely accessing the data such as via the use of an intranet or internet.
The present invention will now be described further and includes other features and analytes that can be included in the methods and apparatus described herein.